Peptide compound, production method therefor, and use thereof

ABSTRACT

Provided are a novel peptide compound, a method of producing the same, and use of the peptide compound. Since the peptide compound has anticancer activity, the peptide compound may be used for the prevention or treatment of cancer.

TECHNICAL FIELD

The present invention relates to a novel peptide compound, a method ofproducing the same, and use of the novel peptide compound.

BACKGROUND ART

Physiologically active substances derived from microorganisms have beena source of antibiotics, antifungal agents, and anticancer drugs, andhave been developed as new drugs for treatment of various diseases orbecome templates for development of new drugs. Examples of antibioticsderived from microorganisms are amphotericin, erythromycin,streptomycin, tetracycline, and vancomycin. In addition, deptomycinisolated from streptomyces which is an actinomyces was approved as anext-generation antibiotic by (FDA) in 2013. Examples of anticancerdrugs originated from microbes are doxorubicin, bleomycin, mithramycin,neocarzinostatin, pentostatin, and epothilone. As such, research onphysiologically active substances derived from bacteria is veryimportant in the development of antibacterial agents, antifungal agents,and antifungal agents.

To screen physiologically active substances that are structurallydifferent from existing substances, one strategy of the recent studiesis to research natural products in a geographically and phylogeneticllyspecific environment. Although readily accessible soil microorganismsand land plants have been extensively studied for natural products overa long period of time, studies on microorganisms or marine origin havebeen relatively inactively done. Oceans cover about 70% of the surfaceof the earth, and the ocean itself is presented as a space ofopportunity that is mostly unexplored. Although the diversity of marinemicroorganisms is not exactly identified, there has been little researchin this area so far that only 1% of marine microorganisms are believedto be cultured or identified.

Therefore, in consideration of the development of structurally newantibiotics and anticancer drugs, it is necessary to select marinemicroorganisms producing useful physiologically active substances, andto explore and develop new compounds producing such marinemicroorganisms.

As such, new strains were discovered while selecting and studying newmarine fungi. In addition, while studying the new strain, it was foundthat the new strain is capable of producing a new peptide compound whichis also accordingly found to exhibit anticancer activity, therebycompleting the present invention.

DETAILED DESCRIPTION OF THE INVENTION Technical Problem

The provided is a novel peptide compound, and an isomer, a derivative,or a pharmaceutically acceptable salt of the peptide compound isomer ofthe peptide compound.

In addition, the provided is a strain F452 of Aspergillus speciesproducing the peptide compound.

In addition, the provided is a method of producing the peptide compound.

In addition, the provided is a pharmaceutical composition for preventingor treating cancer, the pharmaceutical composition including the peptidecompound.

In addition, the provided is a method of preventing or treating cancer.

Technical Solution

According to an aspect, there is provided a novel peptide compoundincluding a lipopeptide and a benzophenone, or an isomer, a derivative,or a pharmaceutically acceptable salt of the peptide compound.

According to another aspect, there is provided a strain F452 ofAspergillus sp. producing the peptide compound.

According to another aspect, there is provided a method of producing thepeptide compound.

According to another aspect, there is provided a pharmaceuticalcomposition comprising the peptide compound or the isomer, thederivative, or the pharmaceutically acceptable salt of the peptidecompound.

According to another aspect, there is provided a method of preventing ortreating cancer, the method using the peptide compound or thederivative, or the pharmaceutically acceptable salt of the peptidecompound.

Advantageous Effects of the Invention

A new peptide including a lipopeptide and a benzophenone has anticanceractivity, and thus, can be used for the prevention or treatment ofvarious cancer types. In addition, a low-priced mass culture medium canbe used to provide a high yield of the peptide compound.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show structure formulas of Asperphenin A and AsperpheninB, respectively;

FIG. 2A is an image showing a medium culturing a strain F452 of thegenus of Aspergillus;

FIG. 3A is a graph showing RKO cell ratios (%) of each cell cycleaccording to the concentration of Asperphenin B, and FIGS. 3B and 3C areeach a graph showing RKO cell ratios (%) of a cell cycle according tothe incubation time of Asperphenin B at a concentration of 2.5 μM and 5μM;

FIG. 4 is a graph showing ratios (%) of living cells, cells of earlyapoptosis, cells of late apoptosis or necrosis, or cells of necrosis,according to the concentration of Asperphenin B;

FIGS. 5A and 5B are each an immunoblot image of cell cycle-relatedproteins according to the concentration of Asperphenin B and theincubation time (in hours) of Asperphenin B at a concentration of 5 μM;

FIGS. 6A and 6B are each an immunoblot image of cell apoptosis-relatedproteins according to the concentration of Asperphenin B and theincubation time (in hours) of Asperphenin B at a concentration of 5 μM;

FIGS. 7A to 7C are each a graph showing cell viability (%) of acombination of Asperphenin B and other anticancer drugs, or otheranticancer drugs only; and

FIG. 8 is a graph showing the tumor volume (mm³) of a mouse according tothe number of days after 4 mg/kg or 8 mg/kg of Asperphenin B wasadministered into the mouse.

BEST MODE

An aspect of the present invention provides a peptide compound includinga lipopeptide and a benzophenone, or an isomer, a derivative, or apharmaceutically acceptable salt of the peptide compound.

The term “peptide compound” as used herein refers to a compoundincluding a peptide. A peptide is a compound in which two or more aminoacids are linked by a peptide bond between a carboxyl group of one aminoacid and an amino group of another amino acid. Depending on the numberof amino acids constituting a peptide, the peptide may be a dipeptide, atripeptide, a tetrapeptide, or the like. A peptide having about 10 orless peptide bonds is called an oligopeptide, and a peptide havingmultiple peptide bonds is called a polypeptide.

The term “isomer” as used herein refers to a compound with the samemolecular formula as another molecule, but with different link orspacial arrangement of constituent atoms in the molecule. The isomer mayinclude, for example, a structural isomer and a stereoisomer.

The term “derivative” as used herein refers to a compound obtained bysubstituting a part of the structure of a compound with another or anatomic group.

The term “pharmaceutically acceptable salt” as used herein refers to aninorganic salt and an organic addition salt of a compound.

The term “lipopeptide” as used herein refers to a substance including alipid connected to a peptide. The lipopeptide may include a lipidconnected to a peptide by an amide bond. The amide bond is also referredto as a peptide bond, and is a covalent bond in which an amino group ofone molecule and a carboxyl group of another molecule are linked.

The lipid may be a substituted or unsubstituted C₁-C₂₀ alkyl group, asubstituted or unsubstituted C₂-C₂₀ alkenyl group, or a substituted orunsubstituted C₂-C₂₀ alkynyl group. The alkyl group may be a C₂-C₂₀alkyl group, a C₅-C₂₀ alkyl group, or a C₁₀-C₁₅ alkyl group. The alkylgroup may include 12 carbons. The alkenyl group may be a C₂-C₂₀ alkenylgroup, a C₅-C₂₀ alkenyl group, a C₁₀-C₂₀ alkenyl group, or a C₁₀-C₁₅alkenyl group. The alkynyl group may be a C₂-C₂₀ alkynyl group, a C₅-C₂₀alkynyl group, a C₁₀-C₂₀ alkynyl group, or a C₁₀-C₁₅ alkynyl group. Theexpression “substituted” as used herein refers means that a hydrogenatom in an organic compound is substituted with another atomic group toform a derivative. Here, a “substituent” is the atomic atom introducedthereto. The substituent may be, for example, a hydroxyl group, ahalogen atom, a C₁-C₂₀ alkyl group, a C₂-C₂₀ alkenyl group, a C₂-C₂₀alkynyl group, a C₁-C₂₀ heteroalkyl group, a C₆-C₂₀ aryl group, a C₆-C₂₀arylalkyl group, a C₆-C₂₀ heteroaryl group, or a C₆-C₂₀ heteroarylalkylgroup, a nitro group, a cyano group, an amino group, an amidino group, ahydrazine group, a hydrazone group, a carboxyl group or a salt thereof,a sulfonic acid or a salt thereof, or a phosphoric acid or a saltthereof.

The peptide may include two, three, or four or more amino acids. Thepeptide may be, for example, a tripeptide including three amino acids.The peptide may include, for example, N terminal-Asparagine(Asp)-Glutamine (Gln)-Leucine (Leu)-C terminal. The peptide may includeone or more beta (β)-amino acids. An amino acid may include an aminogroup, a carboxyl group, and a side chain specific to the amino acid. 20types of standard biological amino acids have an amino group linked to acarbon of a carboxyl group, whereas β-amino acids have an amino grouplinked to β carbon of a carboxyl group. β-amino acids of which sidechains are linked to carbons next to amines are β³-amino acids, andβ-amino acids of which side chains are linked to carbons next tocarboxyl groups are β²-amino acids. The β-amino acid may be β-leucine.The β-amino acid may be β³-leucine.

The benzophenone may be a diphenylmethanone which is an organic compoundhaving a formula of (C₆H₅)₂CO. The benzophenone may be, for example, acompound substituted with 1, 2, or 3 or more hydroxyl groups. Forexample, the benzophenone may be a compound of which at least oneselected from 5^(th) carbon, 9^(th) carbon, and 13^(th) carbon issubstituted with a hydroxyl group.

The lipopeptide and the benzophenone may be linked via a ketone linkage.For example, the benzophenone may be linked to a C-terminal end of thelipopeptide.

The peptide compound may be represented by Formula 1:

In Formula 1, R¹ may be substituted or unsubstituted C₁-C₂₀ alkyl group,a substituted or unsubstituted C₁-C₂₀ alkenyl group, or a substituted orunsubstituted C₁-C₂₀ alkynyl group, wherein R¹ may be selectivelyunsubstituted or substituted with a hydroxyl group,

R², R³, and R⁴ may each independently be selected from hydrogen, ahydroxyl group, a halogen group, a cyano group, —C(═O)R_(a),—C(═O)OR_(a), —OCO(OR_(a)), —C═N(R_(a)), —SR_(a), —S(═O)R_(a),—S(═O)₂R_(a), —PR_(a), a substituted or unsubstituted C₁-C₂₀ alkylgroup, a substituted or unsubstituted C₁-C₂₀ alkoxy group, a substitutedor unsubstituted C₂-C₂₀ alkenyl group, a substituted or unsubstitutedC₂-C₂₀ alkynyl group, a C₂-C₂₀ alkylene oxide group, a substituted orunsubstituted C₃-C₃₀ cycloalkyl group, a substituted or unsubstitutedC₆-C₃₀ aryl group, a substituted or unsubstituted C₆-C₃₀ aryloxy group,a substituted or unsubstituted C₆-C₃₀ heteroaryl group, or a combinationthereof, and

R⁵ may be a substituted or unsubstituted benzophenone.

The alkyl group, the alkenyl group, the alkynyl group, and thesubstitution, and the benzophenone are the same as described above.

The peptide compound may be a compound represented by Formula 2:

The compound represented by Formula may be a compound represented byFormula 3 or 4, or an isomer, a derivative, or a pharmaceuticallyacceptable salt thereof:

An aspect of the present disclosure provides a strain F452 ofAspergillus sp. (Accession Number: KCTC12688BP), the strain producing apeptide compound including a lipopeptide and a benzophenone.

The lipopeptide, the benzophenone, and the peptide compound are the sameas described above.

The strain may include a mutant thereof. The mutant may be, for example,caused by a natural mutant or an artificial mutant. The artificialmutant may be caused by a physical mutagen, such as an ultraviolet ray,or a chemical mutagen, such as base compound.

The strain may include its pore, fungus body, bacterial call, orculture.

The strain may be separated or derived from marine sediments.

An aspect of the present disclosure provides a method of producing apeptide compound including a lipopeptide and a benzophenone, the methodincluding culturing a strain F452 (Accession Number: KCTC12688BP) ofAspergillus sp. to prepare a culture medium; and separating a peptidecompound including a lipopeptide and a benzophenone from the culturemedium.

The method may include culturing the strain F452 of Aspergillus sp.(Accession Number: KCTC12688BP) to prepare a culture medium.

The strain F452 of Aspergillus sp. is the same as described above.

The culturing may be culturing a strain in a liquid medium or a solidmedium.

The medium may include instant ocean. The medium may include carbonsources, such as glucose, rice, starch syrup, dextrin, starch, molasses,animal oils, or plant oils. The medium may include nitrogen sources,such as yeast extracts, peptones, wheat bran, soybean oil meal, wheat,malt, cottonseed meal, fish scrap, corn steep liquor, meat juice,ammonium sulfate, sodium nitrate, or urea. The medium may include, ifnecessary, table salt, potassium, magnesium, cobalt, chlorine,phosphoric acid, sulfuric acid, or inorganic salt that stimulatesproduction of other ions. The medium may include, for example, instantocean, yeast extracts, peptones, and rice.

The culturing may be shake culture or stationary culture that isperformed under aerobic conditions. The temperature at which theculturing is performed may be, for example, in a range of about 20° C.to about 37° C. or about 25° C. to about 30° C., or may be 27° C. Thetime required for the culturing may be, for example 1 day to 2 months, 1week to 2 months, 2 weeks to 2 months, 1 month to 2 months, or 6 weeks.

The method may include separating a peptide compound from the culturemedium, the peptide compound including a lipopeptide and a benzophenone.

The lipopeptide, the benzophenone, and the peptide compound are the sameas described above.

The separating of the peptide compound from the culture medium mayinclude performing concentration, centrifugation, filtration, orchromatography on the culture medium. For example, the culture mediummay be extracted with ethylacetate, water, or a combination thereof. Theobtained concentrate may be divided by chromatography, into 8 fractionsdepending on polarity. The chromatography may be, for example, reversephase flash chromatography using, for example, water, acetonitrile, or acombination thereof as a mobile phase. The fractions may be fractionedby using the reverse phase flash chromatography, to thereby obtain 8fractions.

Among the obtained fractions, fractions eluted with a water/acetonitrilemixed solution, which is mixed at a volume ratio of 50:50, may becapable of separating the peptide compound by using high performanceliquid chromatography (HPLC). The HPLC uses a water/methanol mixedsolution, which is mixed at a volume ratio of 70:30, as a mobile phase,and may be performed by using reversed-phase semi-preparative HPLC. Theseparated peptide compound may have purity of about 80%, about 90%, orabout 99% or more.

An aspect of the present disclosure provides a pharmaceuticalcomposition for preventing or treating cancer, the pharmaceuticalcomposition including a peptide compound including a lipopeptide and abenzophenone, or an isomer, a derivative, or a pharmaceuticallyacceptable salt of the peptide compound.

The lipopeptide, the benzophenone, the peptide compound, the isomer, thederivative, and the pharmaceutically acceptable salt are the same asdescribed above.

The cancer may include, for example, intrahepatic cholangiocarcinoma,liver cancer, thyroid cancer, colon cancer, testis cancer,myelodysplastic syndrome, glioblastoma, oral cavity cancer, mycosisfungoides, acute myeloid leukemia, chronic myeloid leukemia, acutelymphoblastic leukemia, chronic lymphoblastic leukemia, base cellcarcinoma, ovarian epithelial cancer, ovarian germ cell tumor, malebreast cancer, brain tumor, pituitary adenoma, multiple myeloma,gallbladder cancer, biliary tract cancer, colon cancer, retinoblastoma,choroid melanoma, ampullar of vater cancer, bladder cancer, peritonealcancer, parathyroid carcinoma, adrenal gland cancer, non-small cell lungcancer, tongue cancer, astroma, small cell lung cancer, pediatric braintumor, pediatric lymphoma, pediatric leukemia, small bowel neoplasm,meningioma, esophageal cancer, glioma, neuroblastoma, renal pelvis andureter cancer, kidney cancer, malignant soft tissue tumor, malignantbone tumor, malignant lymphoma, malignant mesothelioma, malignantmelanoma, ocular tumor, pudendum cancer, urethral tumor, carcinoma ofunknown primary origin, gastric lymphoma, stomach cancer, gastriccarcinoid tumor, gastrointestinal stromal tumor, Willms tumor, breastcancer, sarcoma, penile carcinoma, pharynx cancer, gestationaltrophoblastic disease, cervical cancer, endometrial cancer, sarcoma ofuterus, prostate cancer, metastatic brain tumor, rectal cancer, rectalcarcinoid tumor, vaginal cancer, spinal tumor, vestibular schwannoma,pancreatic cancer, salivary gland tumor, tonsillar cancer, squamous cellcarcinoma, adenocarcinoma of lung, lung cancer, squamous cell carcinomaof lung, skin cancer, anal cancer, larynx cancer, or a combinationthereof. The cancer may be, for example, lung cancer, colon cancer,stomach cancer, liver cancer, or breast cancer.

The term “prevention” as used herein refers to any action that inhibitsdiseases or delays onset by administration of a composition. The term“treatment” as used herein refers to any action that improves oralleviates a symptom of a disease by the administration of acomposition.

The pharmaceutical composition may further include a known activeingredient having anticancer activity. Such a known active ingredientmay be an anticancer drug. The anticancer drug may be irinotecan,5-fluotouracil, gemcitabine, etoposide, paclitaxel, or a combinationthereof. When the anticancer drug is further included, thepharmaceutical composition may be a single composition or an individualcomposition.

The pharmaceutical composition may further include a carrier, anexcipient, or a diluent. Such a carrier, an excipient, or a diluent maybe, for example, lactose, dextrose, sucrose, sorbitol, mannitol,xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin,calcium phosphate, calcium silicate, cellulose, methylcellulose,microcrystalline cellulose, polyvinylpyrrolidone, water,methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate,or mineral oil.

The pharmaceutical composition may be formulated in the form of oralpreparations such as powders, granules, tablets, capsules, suspensions,emulsions, and syrups, aerosols, external preparations, suppositories,or sterilized injection solutions, according to a conventional method.In the case of formulation, a diluent or an excipient, such as a filler,an extender, a binder, a wetting agent, a disintegrant, and asurfactant, that are typically used may be used.

Regarding the pharmaceutical composition, solid preparations for oraladministration may be tablets, pills, powders, granules, or capsules.The solid preparations may further include an excipient. Such anexcipient may be, for example, starch, calcium carbonate, sucrose,lactose, or gelatin. In addition, the solid preparations may furtherinclude a lubricant, such as magnesium stearate or talc. Regarding thepharmaceutical composition, liquid preparations for oral use may besuspensions, solutions, emulsions, or syrups. The liquid preparationsmay include water or liquid paraffin. The liquid preparations mayinclude an excipient, such as a wetting agent, a sweetening agent, anair freshener, or a preservative. Regarding the pharmaceuticalcomposition, formulations for parenteral administration may besterilized aqueous solutions, non-aqueous solutions, suspensions,emulsions, lyophilized agents, or suppositories. Non-aqueous solutionsor suspensions may include vegetable oils or esters. Vegetable oils mayinclude, for example, propylene glycol, polyethylene glycol, or oliveoil. Esters may include, for example, ethyl oleate. A base of thesuppository may be witepsol, macrogol, tween 61, cacao paper, laurin, orglycerogelatin.

An aspect of the present discloser provides a method of preventing ortreating cancer, the method including administering a pharmaceuticalcomposition for preventing or treating cancer into an individual, thepharmaceutical composition including a peptide compound including alipopeptide and a benzophenone, or an isomer, a derivative, or apharmaceutically acceptable salt of the peptide compound.

The lipopeptide, the benzophenone, the peptide compound, the isomer, thederivative, the pharmaceutically acceptable salt, cancer, prevention,treatment, and the pharmaceutical composition are the same as describedabove.

The individual may be a mammal including a rat, a mouse, a dog, a cow, amonkey, and a human being.

A preferred dosage of the peptide compound varies depending on theconditions and weight of a patient, a degree of disease, drug form, andadministration route and time, but may be appropriately selected by oneof ordinary skill in the art. However, the peptide compound may be, forexample, administered in an amount of about 0.0001 mg/kg to about 100mg/kg, or about 0.001 mg/kg to about 100 mg/kg, once or several times aday. The peptide compound in the pharmaceutical composition may beincluded in an amount of about 0.0001 wt % to about 10 wt % or about0.001 wt % to about 1 wt %, based on the total weight of the totalcomposition.

The pharmaceutical dosage forms of the peptide compound may be in theform of a pharmaceutically acceptable salt of the peptide compound. Thepeptide compound may be used alone or in combination with otherpharmaceutically active compound.

The pharmaceutical composition may be administered in a variety ofroutes to a mammal including a rat, a mouse, a dog, a cow, a horse, amonkey, and a human being. An administration method may be, for example,oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine,or intra-cerebroventricular injections.

The method may further include administering an anticancer drug into anindividual. The peptide compound, its isomer, derivative, orpharmaceutically acceptable salt, and the anticancer drug may beadministered simultaneously, individually, or sequentially. For example,the anticancer drug may be administered into the individual after theadministration of the peptide compound or the isomer, the derivative, orthe pharmaceutically acceptable salt of the peptide compound into theindividual.

Hereinafter, the present disclosure will now be described more fullywith reference to the accompanying Examples below. However, theseExamples are for illustrative purposes only, and should not be construedas being limited to the scope of the inventive concept in any way.

Example 1. Isolation and Identification of Asperphenin a and AsperpheninB

1-1. Isolation of Strain F452 of Aspergillus Species (Aspergillus sp.)

In order to screen a strain producing a substance having anticanceractivity, a strain F452 was isolated from tropical marine sediments. Thewhole genome of the strain F452 was isolated, and an 18S ribosomal DNAsequence was cloned by using a polymerase chain reaction (PCR). Thenucleic acid sequence of the 18S ribosomal DNA was analyzed (SEQ ID NO:1).

As a result of the nucleic acid sequence analysis, the strain F452 wasidentified as a novel strain that was systematically similar toAspergillus versicolor. The strain F452 was named as strain F452 ofAspergillus sp., and then, deposited at the Depositary Authority on Oct.13, 2014 (Accession Number: KCTC12688BP).

1-2. Culture of Strain F452 of Aspergillus sp.

The strain F452 of Aspergillus sp. was inoculated into a sterilized YPGsolid medium (5 g of yeast extract, 5 g of peptone, 10 g of glucose, 16g of agar, and 24.8 g of INSTANT OCEAN® (Aquarium systems) per 1 L ofdistilled water), and then, was subjected to primary culture at atemperature of 27° C. for several days.

The strain F452 cultured in the solid medium of the primary culture wasinoculated into a sterilized YPG liquid medium (5 g of yeast extract, 5g of peptone, 10 g of glucose, and 24.8 g of INSTANT OCEAN® (Aquariumsystems) per 1 L of distilled water), and then, was subjected to secondculture at a temperature of 27° C. for 7 days while being shaken at 150rpm.

10 ml of the second culture was inoculated into a solid rice medium (200g of rice (Organica Co., Ltd., Icheon Rice, Gyeonggi-do), 2.5 g of yeastextract, 2.5 g of peptone, 12.4 g of INSTANT OCEAN® (Aquarium systems)per 500 ml of distilled water), and then, was subjected to third cultureat a temperature of 27° C. for 6 weeks.

1-3. Isolation and Purification of Asperphenin a and Asperphenin B

The solid medium of the third culture in which strain F452 was culturedin Example 1-2 was obtained, and then, 1 L of ethylacetate (DaejungChemicals & Metals Co., Ltd.) was immersed in every 100 g of theobtained solid medium for 1 day. Such a procedure was repeated threetimes in total. Ethylacetate thus obtained was filtered through a filterpaper (Advantec), and the filtrate was decompressed, to thereby removeethylacetate which was a solvent. Such a procedure was repeated toobtain 25 g of a crude extract. 200 ml of methanol (Daejung Chemicals &Metals Co., Ltd.) was added to the crude extract, and then, the methanollayer was decompressed, to thereby obtain 11.4 g of a methanol extract.The methanol extract thus obtained was divided into 8 fractionsaccording to reversed phase chromatography (MERCK, C18, 700 g,reversed-phase). An eluent used in 5 fractions was used by reducingwater by 5% from a water/acetonitril (Burdick & Jackson) solution mixedat a volume ratio of 60:40. The final fraction was divided using 100%methanol (Daejung Chemicals & Metals Co., Ltd.), acetone (DaejungChemicals & Metals Co., Ltd.), and ethylacetate (Daejung Chemicals &Metals Co., Ltd.), to thereby obtain fractions.

Fraction 3 divided using a water/acetonitril solution mixed at a volumeratio of 50:50 was analyzed according to liquid chromatography (LC)-massspectrometry (MS) and based on hydrogen nuclear resonance spectrum. Toidentify the compositions of the fractions, LC/MS using Agilent 1200Series LC (Agilent technologies) and 6130 Series MS was used. Here, themass spectrum was obtained using an LTQ-Orbitrap ESI-MS massspectrometer manufactured by Thermo-Finnigan Company, and wasrepresented in a mass-to-charge (m/z) form. According to the analysis onthe LC-MS and the hydrogen nuclear resonance spectrum, it was confirmedthat the culture medium of the strain contained a novel secondarymetabolite, which was to be named as Asperphenin.

Fraction 3 was isolated using C₁₈ reversed-phase semi-preparative HPLC(particle diameter of 5 μm, 250 mm×10 mm (length×inner particlediameter), elution rate of 2 ml/min) with a refractive index (RI)detector (Shodex). A mobile phase used for the isolation was awater/methanol solution mixed at a volume ratio of 70:30, and thereversed-phase semi-preparative HPLC was performed for about one andhalf an hour. Accordingly, 50.0 mg of Asperphenin A and 46.0 mg ofAsperphenin B were obtained.

1-4. Physicochemical Characterization Analysis of Asperphenin A andAsperphenin B

Asperphenin A and Asperphenin B were in a pale yellow, stable at roomtemperature, and well dissolved in a moderate organic solvent, such asmethanol and acetone. The structures of Asperphenin A and Asperphenin Bwere determined based on nuclear magnetic resonance spectrum, infraredand ultraviolet spectral data, optical rotary power, and high-resolutionmass spectrometry data. The nuclear magnetic resonance spectrum (¹H NMR,¹³C NMR) was obtained using 500 MHz NMR manufactured by Bruker Companyand DMSO-d₆ as a solvent. The mass spectrum was obtained using anLTQ-Orbitrap ESI-MS mass spectrometer manufactured by Thermo-FinniganCompany, and was represented in a mass-to-charge (m/z) form. Theinfrared spectrum was obtained using an FT-IR-4200 spectrometermanufactured by Jasco Company. The ultraviolet spectrum was obtainedusing an U-3010 UV/VIS spectrometer manufactured by Hitachi Company. Theoptical rotary power was obtained using a P-1020 polarimetermanufactured by Jasco Company.

The structure positioning of each of Asperphenin A and Asperphenin B bythe nuclear magnetic resonance spectrum was as follows in Tables 1 and2.

[Asperphenin A]

(1) Molecular formula: C₄₂H₆₁N₅O₁₁

(2) Molecular weight: 811

(3) Color: Pale yellow

(4) Optical rotary power: −24.7 (c 1.0, methanol, 25° C.)

(5) Infrared absorption band (neat): 3309, 1671 wavenumbers

(6) ¹H-NMR (DMSO-d₆, 600 MHZ): see Table 1

(7) ¹³C-NMR (DMSO-d₆, 150 MHZ): see Table 1

TABLE 1 Chemical shift values of nuclear magnetic resonance spectrum ofAsperphenin A mult (J Position δ_(c) Type δ_(H) in Hz)  1 136.0 C  2120.4 CH 7.21 s  3 138.5 C  4 120.4 CH 6.84 s  5 153.4 C  6 128.6 C  7201.8 C  8 111.1 C  9 161.6 C 10 106.8 CH 6.19 d (8.3) 11 135.7 CH 7.15t (8.3) 12 106.8 CH 6.19 d (8.3) 13 161.6 C 14 20.8 CH₃ 2.29 s 15 198.3C 16 44.7 CH₂ 3.00 dd (16.5, 4.5) 2.85 dd (16.5, 8.7) 17 43.4 CH 4.13 m17- 7.66 d (8.3) NH 18 42.5 CH₂ 1.28 m 0.98 ddd (13.7, 9.4, 3.4) 19 24.1CH 1.45 m 20 21.3 CH₃ 0.66 d (6.7) 21 23.3 CH₃ 0.71 d (6.7) 22 170.2 C23 52.6 CH 4.02 m 23- 7.92 d (7.7) NH 24 27.6 CH₂ 1.87 m 1.65 m 25 31.4CH₂ 2.02 t (7.9) 26 173.9 C 26- 7.14 br s NH₂ 6.70 br s 27 170.9 C 2849.8 CH 4.44 m 28- 8.04 d (7.5) NH 29 36.9 CH₂ 2.40 m 30 171.8 C 30-7.39 br s NH₂ 6.91 br s 31 171.2 C 32 43.7 CH₂ 2.18 m 33 67.5 CH 3.75 m33- 4.59 brs OH 34 37.0 CH₂ 1.30 m 35 25.1 CH₂ 1.30 m 1.19 m 36 31.3 CH₂1.19 m 37 29.1 CH₂ 1.19 m 38 29.1 CH₂ 1.19 m 39 29.0 CH₂ 1.19 m 40 28.7CH₂ 1.19 m 41 22.1 CH₂ 1.21 m 42 13.9 CH₃ 0.81 t (7.0)

[Asperphenin B]

(1) Molecular formula: C₄₂H₆₁ N₅O₁₁

(2) Molecular weight: 811

(3) Color: Pale yellow

(4) Optical rotary power: −18.4 (c 1.0, methanol, 25° C.)

(5) Infrared absorption band (neat): 3309, 1671 wavenumbers

(6) ¹H-NMR (DMSO-d₆, 600 MHZ): see Table 2

(7) ¹³C-NMR (DMSO-d₆, 150 MHZ): see Table 2

TABLE 2 Chemical shift values of nuclear magnetic resonance spectrum ofAsperphenin B mult (J Position δ_(c) Type δ_(H) in Hz)  1 136.0 C  2120.3 CH 7.21 s  3 138.6 C  4 120.5 CH 6.84 s  5 153.4 C  6 128.7 C  7201.8 C  8 111.1 C  9 161.6 C 10 106.8 CH 6.19 d (8.1) 11 135.7 CH 7.14t (8.1) 12 106.8 CH 6.19 d (8.1) 13 161.6 C 14 20.8 CH₃ 2.29 s 15 198.4C 16 44.6 CH₂ 2.97 dd (16.4, 4.2) 2.83 dd (16.4, 8.5) 17 43.5 CH 4.13 m17- 7.63 d (8.2) NH 18 42.6 CH₂ 1.34 m 1.01 m 19 24.2 CH 1.46 m 20 21.4CH₃ 0.69 d (6.4) 21 23.4 CH₃ 0.74 d (6.4) 22 170.4 C 23 52.7 CH 4.02 m23- 8.03 d (8.4) NH 24 27.5 CH₂ 1.87 m 1.65 m 25 31.5 CH₂ 2.02 m 26174.1 C 26- 7.16 br s NH₂ 6.74 br s 27 171.0 C 28 49.9 CH 4.46 ddd (6.9,6.9, 5.1) 28- 8.05 d (5.1) NH 29 37.0 CH₂ 2.53 dd (15.6, 6.9) 2.42 dd(15.6, 6.9) 30 171.9 C 30- 7.41 br s NH₂ 6.95 br s 31 171.3 C 32 43.7CH₂ 2.18 m 2.18 m 33 67.5 CH 3.75 m 33- 4.60 brs OH 34 37.0 CH₂ 1.30 m35 25.1 CH₂ 1.30 m 1.19 m 36 31.3 CH₂ 1.19 m 37 29.1 CH₂ 1.19 m 38 29.1CH₂ 1.19 m 39 29.0 CH₂ 1.19 m 40 28.8 CH₂ 1.19 m 41 22.1 CH₂ 1.22 m 4214.0 CH₃ 0.81 t (6.7)

The structure of each of Asperphenin A and Asperphenin B analyzed on thebasis of the nuclear magnetic resonance spectrum was represented by achemical formula below.

Example 2. Anticancer Activity of Asperphenin A and Asperphenin B

2-1. Verification of Anticancer Activity of Asperphenin A andAsperphenin B

Regarding a lung cancer cell line A549 (Korean Cell line Bank), a coloncancer cell line HCT116 (ATCC), a stomach cancer cell line SNU638(Korean Cell Line Bank), a liver cancer cell line SK-HEP-1 (Korean Cellline Bank), and a breast cancer cell line MDA-MB-231 (Korean Cell lineBank), a sulforhodamine B (SRB) assay which is a method of measuringcell viability was used for measuring apoptosis effects of Asperphenin.

In detail, 190 μl of a cell suspension at a concentration of 3.5×10⁴cells/ml was inoculated in each well of a 96-well microplate. 0.8 μM, 4μM, 20 μM, or 100 μM of Asperphenin was added to the cell culturemedium, and cultured at a temperature of 37° C. for 72 hours under thecondition of 5% CO₂. After incubation, 50 μl of 50% (v/v)trichloroacetic acid solution (Sigma Aldrich) was added to each well,and then, incubated at a temperature of 4° C. for 30 minutes, to therebyfix the cells thereto. The fixed cells were washed with water fivetimes, and then, dried in the air.

Next, 80 μl of a 0.4% (w/v) SRB aqueous solution (Sigma Aldrich)containing 1% (v/v) acetic acid (DUKSAN) was added to each well,incubated at room temperature for 1 hour to stain cells therein, and thestained cells washed with water and dried. The cells were dissolved byadding 200 μl of 10 mM Tris (pH 10.0) (Sigma Aldrich) to each well, andthen, the number of living cells was calculated by measuring theabsorbance thereof at 515 nm. The concentration of the compoundinhibiting cell growth to 50% based on the number of the living cells,i.e., 50% inhibition concentration (IC₅₀) was calculated, and theresults are shown in Table 3. Here, as a positive control, etoposide(Sigma Aldrich) was used.

TABLE 3 Cell growth inhibition concentrations of Asperphenin A andAsperphenin B (IC₅₀, μM) MDA-MB- A549 HCT116 SNU638 SK-HEP-1 231Asperphenin A 14.6 1.7 5.8 2.3 3.1 Asperphenin B 41.8 2.6 11.7 3.0 6.0Etoposide 0.7 1.9 0.8 0.6 10.6

As shown in Table 3, Asperphenin A exhibited strong cell inhibitioneffects against the cell lines of lung cancer, colon cancer, stomachcancer, and breast cancer. Asperphenin B exhibited strong cellinhibition effects against the cell lines of colon cancer, stomachcancer, liver cancer, and breast cancer, except for the cell line A549.In particular, Asperphenin A and Asperphenin B both exhibited similaranticancer activity to or better anticancer activity against the coloncancer cell line HCT116 than that of etoposid which is a positivecontrol.

2-2. Verification of Anticancer Activity of Asperphenin B Against ColonCancer Cell Line

As shown in Table 3, it was confirmed whether Asperphenin B had cellgrowth inhibition effects against not only the colon cancer cell lineHCT116, but also other colon cancer cell lines.

From the colon cancer cell lines HCT116 (ATCC), HCT15 (Korean Cell LineBank), LS174T (Korean Cell Line Bank), RKO(ATCC), and SW480(ATCC), cellgrowth inhibition concentrations (IC₅₀, μM) were calculated as describedin Example 2-1, and the results are shown in Table 4. Here, as apositive control, paclitaxel (Sigma Aldrich) was used.

TABLE 4 Growth inhibition value of cancer cell against Asperphenin B(IC₅₀) HCT15 HCT116 LS174T RKO SW480 Asperphenin B (μM) 7.20 4.05 1.841.17 31.35 Paclitaxel (nM) >100 0.42 0.46 0.21 >100

As shown in Table 4, Asperphenin B strongly inhibited the cell growth of4 colon cancer cell lines, except for the cell line SW480, and moreparticularly, Asperphenin B exhibited the strongest inhibitory effectsagainst the cell line RKO.

2-3. Measurement of Cell Cycle Change in Colon Cell Line by AsperpheninB

The effects of Asperphenin B on the cell cycle of the colon cancer cellline RKO was confirmed by flow cytometry.

RKO cells (ATCC) were diluted in a medium containing 10% (v/v) FBS tobecome 1×10⁵ cells/ml, and then, inoculated into a 60 mm culture dish.The inoculated cells were cultured for about 24 hours at a temperatureof 37° C. under the condition of 5% CO₂. The cultured cells were washedwith phosphate-buffered saline (PBS) once, and the medium was replacedby a fresh medium. Asperphenin B at the final concentration of 0.625 μM,1.25 μM, 2.5 μM, 5 μM, or 10 μM was added to the cultured cells, andthen, the cells were cultured at a temperature of 37° C. under conditionof 5% CO₂.

After a certain period of time, cells attached to the culture dish andcells not attached to the medium were collected. The collected cells waswashed with PBS once, and then, 1 ml of cold 70% (v/v) ethanol. Thecells were incubated at a temperature of 4° C. for about 12 hours to fixthe cells. After removing 70% (v/v) ethanol, the fixed cells were washedwith PBS once. 500 μl of RNase A (Sigma Aldrich) at a concentration of50 μg/ml as added to the cells, and the cells were incubated at roomtemperature for about 30 minutes. Propidium iodide (PI) at a finalconcentration of 50 μg/ml was added to the cells, and then, the cellswere incubated at room temperature for about 30 minutes in a conditionwhere reactants were in the shaded state. The cells stained with PI weresubjected to analysis of cell cycle by using a BD FACSCalibur flowcytrometer (manufactured by BD Biosciences). Based on the flow cytometryresults, cell ratios at the sub-G₁, G₀/G₁, S, and G₂/M phases werecalculated. Here, as a negative control, cells to which Asperphenin Bwas not added was used.

When the RKO cells were incubated for 48 hours in the presence ofAsperphenin B, cell ratios (%) dependent on the concentration ofAsperphenin B are shown in FIG. 3A and Table 5. When the RKO cells wereincubated in the presence of Asperphenin B at the concentrations of 2.5μM and 5 μM, cell ratios (%) dependent on the incubation time are shownin FIGS. 3B and 3C, and Table 6.

TABLE 5 Asperphenin B concentrations Cell 0 μM cycle (negative phasecontrol) 0.625 μM 1.25 μM 2.5 μM 5 μM 10 μM Sub-G₁ 3.03% 7.14% 24.18%38.60% 59.90% 67.82% G₀/G₁ 59.69% 52.49% 23.02% 17.99% 17.59% 17.07% S14.85% 8.11% 6.39% 6.63% 5.29% 5.46% G₂/M 20.50% 29.56% 43.94% 34.58%15.97% 8.92%

TABLE 6 Cell 2.5 μM Asperphenin B 5 μM Asperphenin B cycle Control 6 1224 48 6 12 24 48 phase 0 hour hours hours hours hours hours hours hourshours Sub-G₁ 4.40% 3.77% 3.25% 5.03% 33.38% 3.19% 3.80% 4.86% 48.23%G₀/G₁ 66.21% 62.16% 56.50% 30.73% 17.80% 60.66% 57.60% 28.35% 20.53% S9.70% 13.82% 15.09% 17.38% 6.55% 13.48% 14.63% 14.08% 5.90% G₂/M 17.55%18.28% 22.18% 40.76% 39.31% 20.21% 20.93% 48.88% 23.37%

As shown in FIG. 3A and Table 5, in the case where the RKO cells andAsperphenin B were incubated for 48 hours, the cells at the sub-G₁ phasewere increased according to the concentrations of Asperphenin, ascompared to the control, whereas the cells at the G₀/G₁ and S phaseswere decreased. The cells in the G₂/M phase were increased as comparedto a control in which low-concentrated Asperphenin B was treated, andwere decreased as compared to a control in which high-concentratedAsperphenin B was treated. In addition, as shown in FIGS. 3B and 3C, andTable 6, the cells in the sub-G₁ and G₂/M phases were changed dependingon the treatment time of Asperphenin B. In the case where Asperphenin Bat the concentration of 2.5 μM or 5 μM was treated for about 24 hours,the cells in the G₂/M phase increased as compared to a control. In thecase Asperphenin B at the concentration of 2.5 μM or 5 μM was treatedfor about 48 hours, the cells in the G₂/M phase decreased as compared tothe case where Asperphenin B at the concentration of 2.5 μM or 5 μM wastreated for about 24 hours. The ratio of cells in the sub-G₁ phaseshowing no change for 0 to 24 hours during the treatment of AsperpheninB, increased. It was confirmed that the cells in the G₂/M phase occurredcell cycle arrest until 24 hours of the treatment of Asperphenin B, butafter 48 hours of the treatment of Asperphenin B, the cell apoptosis wasinduced.

2-4. Measurement of Induction of Apoptosis by Asperphenin B

The process of apoptosis in the cell line RKO by Asperphenin B wasconfirmed using a Annexin V-FITC apoptosis measuring kit (manufacturedby D Pharmingen).

As described in Example 2-3, Asperphenin B at the concentration of 0.625μM, 1.25 μM, 2.5 μM, 5 μM, or 10 μM was added to the RKO cells, culturedfor 48 hours to obtain cells. 300 μl of 1× binding buffer was added tothe obtained cells, and well mixed together. Then, 5 μl of Annexin V and5 μl of PI were added to 100 μl of the cell mixture, and allowed toreact at room temperature for 15 minutes in the shaded stated. 400 μl of1× binding buffer was added to the reactants, a FACSCalibur flowcytometer (BD FACSCalibur, BD Biosciences) was used to analyze the cellapoptosis. Based on the flow cytometry results, ratios of cells thatwere not stained (i.e., living cells), cells stained with annexin V(i.e., cells apoptosized at the early state), cells stained with both PIand annexin V (i.e., cells apoptosized at the late state or cellsnecrotized,), or cells stained with PI only (i.e., cells necrotized)were calculated. As a negative control, cells to which Asperphenin B wasnot added were used.

When the RKO cells were incubated for 48 hours in the presence ofAsperphenin B, cell ratios (%) depending on the concentrations ofAsperphenin B are shown in FIG. 4. As shown in FIG. 4, the ratios of thecells of early apoptosis and the cells of late apoptosis or necrosisincreased as compared to the ratio of the control. Here, the ratio ofnecrotic cells was increased as compared to that of the control.Therefore, it was confirmed that Asperphenin B induced apoptosis andnecrosis of the RKO colon cancer cell line.

2-5. Evaluation of Effects of Asperphenin B on Cell Cycle-RelatedProteins and Apoptosis-Related Proteins

As described in Example 2-3, cells were obtained in a way thatAsperphenin B at the concentration of 0.625 μM, 1.25 μM, 2.5 μM, 5 μM,or 10 μM was added to the RKO cells, and then, cultured for 48 hours.However, cells were obtained in a way that Asperphenin B at theconcentration of 5 μM was added to the RKO cells, and then, cultured for0 to 48 hours. Then, proteins were obtained from the obtained cells.

Regarding the expression of the cell cycle-related proteins,anti-p-cyclin B1(Ser147) antibody (Cell Signaling Technology),anti-cyclin B1 antibody (Santa Cruz), anti-p-cdc2(Tyr15) antibody (CellSignaling Technology), anti-cdc2 antibody (Santa Cruz), and anti-β-actinantibody (Santa Cruz) were used to perform immunoblotting thereon.Images obtained by immunoblotting are shown in FIGS. 5A and 5B.

As shown in FIG. 5A, when the RKO cells were incubated for 48 hours inthe presence of Asperphenin B, the expression of non-active p-cdc2(Tyr15) protein was increased in a concentration-dependent manner. Theexpression of non-active p-cyclin B1 (Ser147) protein was increased inthe cells treated with 2.5 μM of Asperphenin B. The expression ofp-cyclin B1 (Ser147) protein and cyclin B1 protein decreased in thecells treated with 5 μM and 10 μM of Asperphenin B. In addition, asshown in FIG. 5B, when the cells were treated with 5 μM of AsperpheninB, the expression of p-cdc2 (Tyr15) protein was increased intime-dependent manner, and the expression of p-cyclin B1 (Ser147) washighest at about 24 hours. Therefore, it was confirmed that AsperpheninB regulated the expression of cell cycle-related factors in the RKOcolon cancer cells.

Also, regarding the expression of apoptosis-related proteins, anti-ATMantibody (Cell Signaling Technology), anti-p-Chk (Thr68) antibody (CellSignaling Technology), anti-Chk antibody (Cell Signaling Technology),anti-p-H2AX antibody (Cell Signaling Technology), anti-p53 antibody(Santa Cruz), anti-Bax antibody (Santa Cruz), anti-BID antibody (CellSignaling Technology), anti-Caspase-8 antibody (Cell SignalingTechnology), anti-Caspase-3 antibody (Cell Signaling Technology),anti-Caspase-9 antibody (Cell Signaling Technology), anti-cleaved PARPantibody (BD Biosciences), and anti-β-actin antibody (Santa Cruz) wereused, to thereby perform immunoblotting. Images obtained byimmunoblotting are shown in FIGS. 6A and 6B.

As shown in FIG. 6A, when the RKO cells were incubated for 48 hours inthe presence of Asperphenin B, the expression of ATM protein wasincreased in a concentration-dependent manner while Chk2 and H2AX, whichare sub-regulatory factors of ATM proteins, were phosphorylated to beactivated. The expression of p53 protein was increased in aconcentration-dependent manner, leading to the increased expression ofBax. In addition, the cleavage of poly(ADP-ribose) polymerase (PARP),which is a substrate of Bid, caspase-8, caspase-9, caspase-3, andcaspase-3, have been induced by Asperphenin B. As shown in FIG. 6B, whenthe cells were treated with 5 μM of Asperphenin B, the expression ofapoptosis-inducing proteins and marker proteins, such as p-Chk2 (Thr68),p-H2AX (Ser139), cleaved caspase-8, cleaved caspase-9, cleavedcaspase-3, and PARP, was increased in a group where the cells weretreated for 48 hours. The expression of p53 and Bax was increased in atime-dependent manner. Accordingly, it was confirmed that Asperphenin Bregulated the expression of apoptosis-related factors of the RKO coloncancer cells.

2-6. Production of Reactive Oxygen Species (ROS) by Asperphenin B

ROS was capable of inducing apoptosis within a cell, and in this regard,it was examined whether ROS has been produced within a cell byAsperphenin B.

In detail, as described in Example 2-3, Asperphenin B at theconcentration of 2.5 μM, 5 μM, or 10 μM wad added to the RKO cells.Cells were collected after the RKO cells were cultured for 24 hours. Inaddition, a cell group in which 5 mM of N-acetylcysteine (NAC) (SigmaAldrich), which is an antioxidant, was added to 5 μM and 10 μM ofAsperphenin B was prepared as a comparative group, whereas a cell groupin which none of Asperphenin B and NAC was contained as a control.

Afterwards, 2′,7′-dichlorofluorescin diacetate (DCFH-DA) (Sigma Aldrich)having a final concentration of 20 μM was added to the cell culturemedium, and then, cultured at a temperature of 37° C. for 30 minutesunder the condition of 5% CO₂. Cells attached to the cell medium andcells not attached to the cell medium were all collected, washed withcold PBS twice, and then, suspended again in 1 ml of PBS to measureintensity of 2′,7′-dichlorofluorescin (DCF) by using a BD FACSCaliburflow cytrometer (manufactured by BD Biosciences). Based on the intensityof DCF, ratios of cells producing ROS were calculated, and resultsthereof are shown in table 7.

TABLE 7 Drug Ratio of cells producing ROS No drug treatment (control)5.95% 2.5 μM Asperphenin B 8.14%   5 μM Asperphenin B 13.86%  10 μMAsperphenin B 14.26%   5 μM Asperphenin B + 5 mM NAC 1.41%  10 μMAsperphenin B + 5 mM NAC 2.55%

As shown in Table 7, when the RKO cells were incubated for 24 hours inthe presence of Asperphenin B, the production of ROS increased about 2.4times as much as the production of ROS in the control. However, theproduction of ROS by Asperphenin B was inhibited by NAC, which is anantioxidant. Therefore, it was confirmed that Asperphenin B induced theproduction of ROS in the RKO colon cancer cells.

2-7. Administration of Asperphenin B in Combination with OtherAnticancer Drugs

The effects of administration of Asperphenin B in combination with otheranticancer drugs were examined in vitro.

RKO cells diluted in a medium containing 10% (v/v) FBS were inoculatedin each well of a 96-well microplate so that each plate included 7×10³cells, and then, the cells were cultured for about 24 hours at atemperature of 37° C. under the condition of 5% CO₂. In the mediumcontaining 10% (v/v) FBS, irinotecan (Sigma Aldrich), 5-fluorouracil(Sigma Aldrich), or gemsitabin (Sigma Aldrich) was mixed withAsperphenin B at a ratio of 1:1, and then, the mixture was added to thecultured cells. Then, the cells were cultured for 48 hours at atemperature of 37° C. under the conditions of 5% CO₂. The cell viabilitywas measured according to an SRB assay, and the measured cell viabilityresults are shown in FIGS. 7A to 7C. FIG. 7A shows the cell viability(%) in the case using irinotecan only at the concentrations of 1.25 μMto 10 μM (●) and the case using irinotecan and 2.5 μM of Asperphenin Bin combination (▪). FIG. 7B shows the cell viability (%) in the caseusing gemsitabin only at the concentrations of 1 nM to 100 nM (●) or thecase using gemsitabin and 8 μM of Asperphenin B in combination (▪). FIG.7C shows the cell viability (%) in the case using 5-fluorouracil at theconcentrations of 0.1 μM to 10 μM (●) or 5-fluorouracil and 8 μM ofAsperphenin B in combination (●).

In addition, the effects of the administration in combination weremeasured according to Equation 1, and results thereof are shown in Table8.Effects of administration in combination=D1/(Dx)1+D2/(Dx)2  [Equation 1]

D1: Concentration of Asperphenin B with expected effect inadministration in combination

D2: Concentration of other anticancer drugs with expected effect inadministration in combination

(Dx)1: Concentration of Asperphenin B with expected effect inadministration of Asperphenin B only

(Dx)2: Concentration of other anticancer drugs with expected effect inadministration of other anticancer drugs only

When the calculated effect of the administration in combination is <1,=1, and >1, it is meant to be synergistic effect, additive effect, andantagonistic effect, respectively.

TABLE 8 Level symbols Conc. of Conc. of Effect of representinganticancer Asperphenin administration effect of Anticancer drug B inadministration drug (μM) (μM) combination in combination Irinotecan 1.252.5 0.792 ++ Irinotecan 2.5 2.5 0.751 ++ 5 2.5 0.691 +++ 10 2.5 0.650+++ 5- 0.08 8 0.863 + fluorouracil 5- 0.4 8 0.852 + fluorouracil 2 80.786 ++ 10 8 0.765 ++ Gemsitabin 0.32 8 0.907 ± (μM) 1.6 8 0.882 + 8 80.801 ++ 40 8 0.714 ++

As shown in FIGS. 7A to 7C and Table 8, the effects of cell growthinhibition increased in the case where Asperphenin B was administratedin combination, as compared to the case where irinotecan,5-fluorouracil, or gemsitabin was administered alone.

2-8. Verification of Anticancer Effect of Asperphenin B in TumorXenograft Mouse Model

The anticancer effect of Asperphenin B was verified in a tumor xenograftmouse model to which human colon cancer cell line was transplanted.

In detail, RKO cells were subcutaneously injected at a concentration of3.5×10⁶ cells/150 μl into the right side of a nude mouse (Central Lab.Aminol Inc., hairless and small mouse that was tymus free and had driedbeard at birth). When the tumor size reached 60 mm³ after 14 days of theinjection into the RKO cells, 4 mg/kg or 8 mg/kg of Asperphenin B wasadministered intraperitoneally 3 times a week, i.e., a total of 21 days(n=5). The tumor size was measured by using a digital caliper for 21days at intervals of 3 to 4 days. Here, as a control, a nude mouse towhich Asperphenin B was not administered was used (n=5).

A tumor volume was calculated according to Equation 2, and a tumorgrowth inhibition ratio was calculated according to Equation 3 based onthe calculated tumor volume.Tumor volume (mm³)=(length)×(width)×(height)×π/6  [Equation 2]Tumor growth inhibition ratio (%)=[1−(final mean tumor volume inAsperphenin B-treated group)/(final mean tumor volume incontrol)]×100  [Equation 3]

After administration of Asperphenin B, the tumor volume (mm³) of themouse according to the number of days are shown in FIG. 8 (●: control,●: administration of 4 mg/kg of Asperphenin B, ▴: administration of 8mg/kg of Asperphenin B, *: p<0.05, **: p<0.01, ***: p<0.005), and thetumor growth inhibition ratio calculated is shown in Table 9.

TABLE 9 Administration group 4 mg/kg of Asperphenin B 8 mg/kg ofAsperphenin B Inhibition ratio 38.9 68.7 (%)

As shown in Tables 8 and 9, the inhibition of tumor growth was observedin a compound concentration-dependent manner in the group administeredwith Asperphenin B.

Name of Depositary Authority: Collection of Microorganisms(International)

Accession Number: KCTC12688BP

Accession date: 20141013

The invention claimed is:
 1. A pharmaceutical composition comprising apeptide compound represented by Formula 1 or the isomer, the derivative,or the pharmaceutically acceptable salt of the peptide compound, and ananticancer drug;

In Formula 1, R¹ is a substituted or unsubstituted C₁-C₂₀ alkyl group, asubstituted or unsubstituted C₁-C₂₀ alkenyl group, or a substituted orunsubstituted C₁-C₂₀ alkynyl group, wherein R¹ is selectivelyunsubstituted or substituted with a hydroxyl group, R² and R³ are eachindependently a C₁-C₂₀ alkyl group substituted with —C(═O)NH₂, R⁴ ishydrogen, a hydroxyl group, a halogen group, a cyano group, asubstituted or unsubstituted C₁-C₂₀ alkyl group, a substituted orunsubstituted C₁-C₂₀ alkoxy group, a substituted or unsubstituted C₂-C₂₀alkenyl group, a substituted or unsubstituted C₂-C₂₀ alkynyl group, aC₂-C₂₀ alkylene oxide group, a substituted or unsubstituted C₃-C₃₀cycloalkyl group, a substituted or unsubstituted C₆-C₃₀ aryl group, asubstituted or unsubstituted C₆-C₃₀ aryloxy group, a substituted orunsubstituted C₆-C₃₀ heteroaryl group, or a combination thereof, and R⁵is a substituted or unsubstituted benzophenone.
 2. The pharmaceuticalcomposition of claim 1, wherein the peptide compound is represented byFormula 2:


3. The pharmaceutical composition of claim 2, wherein the compoundrepresented by Formula 2 is represented by Formula 3 or 4:


4. The pharmaceutical composition of claim 1, wherein the pharmaceuticalcomposition is for preventing or treating cancer.
 5. The pharmaceuticalcomposition of claim 4, wherein the cancer is lung cancer, colon cancer,stomach cancer, liver cancer, or breast cancer.
 6. The pharmaceuticalcomposition of claim 1, wherein the anticancer drug is irinotecan,5-fluorouracil, gemsitabin, etoposide, paclitaxel, or a combinationthereof.
 7. The pharmaceutical composition of claim 1, wherein thepharmaceutical composition is a single composition or a separatecomposition.